LOCUS       sequence-275600-        6437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GST tagged AO7 encoding aa 126-258 with a P199A mutation for 
            expression in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6437)
  AUTHORS   Li S, Liang YH, Mariano J, Metzger MB, Stringer DK, Hristova VA, Li 
            J, Randazzo PA, Tsai YC, Ji X, Weissman AM
  TITLE     Insights into Ubiquitination from the Unique Clamp-like Binding of 
            the RING E3 AO7 to the E2 UbcH5B.
  JOURNAL   J Biol Chem. 2015 Dec 18;290(51):30225-39. doi: 
            10.1074/jbc.M115.685867. Epub 2015 Oct 16.
  PUBMED    26475854
REFERENCE   2  (bases 1 to 6437)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M115.685867"; journalName: "J Biol Chem"; date: 
            "2015-12-18- 18"; volume: "290"; issue: "51"; pages: "30225-39"
COMMENT     Sequence Label: pETDUET-GST AO7RE P199A
FEATURES             Location/Qualifiers
     source          1..6437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        141..159
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    160..184
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             199..221
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             228..884
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MGSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     842..864
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             894..917
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        1391..1409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1410..1434
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             1543..1587
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1625..1643)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1639..1686
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1723..2178
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1810..1829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2020..2041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2293..3084,3085..3153))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2916..2935
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3154..3246)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3327..3915
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3816..3835
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4069..4086
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4101..4240
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4166..4185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4326..4348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4342..4533)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5105..6187)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6149..6168
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6188..6265)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc
       61 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
      121 cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      181 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggctccccta
      241 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      301 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      361 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      421 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      481 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      541 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      601 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      661 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      721 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      781 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      841 agggctggca agccacgttt ggtggtggcg accatcctcc aaaacaggat ccgctggaag
      901 ttctgttcca ggggcccaca gataacaaca tccctcatgg ccagtgtgtc atctgcctct
      961 atggtttcca ggagaaggag gcctttacca aaacaccctg ttaccactac ttccactgcc
     1021 actgccttgc tcggtacatc cagcacatgg agcaagagct gaaggcacaa ggacaggagc
     1081 aggaacagga acggcagcat gctacaacca aacagaaggc agtcggtgtg cagtgtgcag
     1141 tgtgcagaga gcccctcgtg tatgatcttg cctcactgaa agcagcccct gaaccccaac
     1201 agcccatgga gctgtaccag cccagtgcag agagcttgcg ccagcaagaa gaacgcaagc
     1261 ggctctacca gaggcagcag gagcgggggg gaatcattga ccttgaggct gagcgataga
     1321 agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta cacggccgca
     1381 taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccatct
     1441 tagtatatta gttaagtata agaaggagat atacatatgg cagatctcaa ttggatatcg
     1501 gccggccacg cgatcgctga cgtcggtacc ctcgagtctg gtaaagaaac cgctgctgcg
     1561 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     1621 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1681 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1741 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1801 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1861 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1921 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1981 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2041 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2101 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2161 tattaacgtt tacaatttct ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac
     2221 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2281 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2341 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2401 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2461 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2521 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2581 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2641 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2701 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2761 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2821 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2881 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2941 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3001 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3061 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3121 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat catgattgaa
     3181 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3241 aacaaatagg tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3301 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3361 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3421 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3481 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3541 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3601 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3661 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3721 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3781 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3841 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3901 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3961 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4021 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4081 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4141 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4201 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4261 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4321 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4381 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4441 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4501 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4561 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4621 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4681 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4741 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4801 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4861 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4921 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4981 cacgatcatg ctagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5041 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5101 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5161 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5221 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5281 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5341 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     5401 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5461 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5521 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5581 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5641 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5701 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5761 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5821 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5881 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5941 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6001 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6061 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6121 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6181 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6241 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6301 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6361 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6421 ccgaaacaag cgctcat
//
